The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular diseases, obesity, and diabetes. Medications targeted to PPARs have been established to treat hyperlipidemia (fibrates) and insulin resistance (glitazones). Thus, there is significant interest in developing new and specific ligands for these receptors. Here, we present the results of the screening of new ligands of PPARalpha and PPARgamma. Optical isomers of new chiral fibrates were synthesized and tested in cell-based assays. Compound (S)-7 showed a dual PPARalpha/gamma activity, and its stereochemistry was crucial in receptor activation. Protease protection experiments suggested that this compound binds directly to PPAR. Moreover, computational studies showed that it properly docks to PPARalpha and gamma ligand binding pockets. Interestingly, (S)-7 exhibited only a modest capacity to induce the differentiation of murine fibroblasts 3T3-L1 into adipocytes compared to rosiglitazone, a well-known PPARgamma agonist.

CLC-K ClϪ channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-offunction mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 M range.Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLCKb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site.

No abstract

ClC-Ka and ClC-Kb Cl ؊ channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 M). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxypropionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 M, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.kidney ͉ molecular modeling ͉ pharmacology

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPAR␥, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal struc- The peroxisome proliferator-activated receptors (PPARs), 4 members of the superfamily of nuclear receptors, are eukaryotic transcription factors that control the expression of genes involved in fatty acid metabolism (1, 2). They function as cellular lipid sensors that activate transcription in response to the binding of a cognate ligand, generally fatty acids and their eicosanoids metabolites (3). Ligand binding, by promoting the stabilization of the active conformation of the C-terminal activation function-2 helix (H12), triggers the recruitment of co-activator proteins (1, 4, 5) that locally remodel chromatin and activate the cellular transcriptional machinery (6, 7). The subtype PPAR␣ promotes fatty acid catabolism in the liver and skeletal muscle, whereas PPAR␥ regulates fatty acid storage in adipose tissues (1). PPARs are also targets of synthetic hypolipidemic and antidiabetic drugs. In particular, the fibrate class of lipid-lowering drugs (e.g. fenofibrate and gemfibrozil) are PPAR␣ ligands (8 -10). The thiazolidinedione (11, 12) antidiabetic agents (rosiglitazone and pioglitazone) are PPAR␥ agonists whose insulin-sensitizing action is well established (13). Selective PPAR␥ modulators (1, 14), acting in a cell type-selective manner and having lower adverse effects, such as edema, cardiomegaly, increased adiposity, and weight gain (14 -16), have been identified. These ligands, which may function as partial agonists, provide diminished conformational stability of receptor as opposed to full agonists (1, 5, 17). Furthermore, PPAR ligands with a dual activity on both PPAR␣ and PPAR␥ receptors improve hyperglycemia and dyslipidemic disorders in a coordinate manner (1). Such compounds may lead to preferred therapies for diabetes, obesity, or metabolic syndrome. For this reason, the search for dual agonists with lower potency on PPAR␥ as compared with full agonists, but also active on PPAR␣, would be desirable.We synthesized the two enantiomers of the novel compound, 2-(4-{2-[1,3-benzoxazol-2-yl(heptyl)amino]ethyl}phenoxy)-2-methyl-butanoic acid, a conformationally constrained analogue of the well known PPAR␣/␥ agonist GW2331 (3, 18) (Fig. 1). The R-enantiomer, (R)-1, is able to activate both PPAR␣ and * This work was supported by Italian Ministry of University and ResearchGrant Progetti di Ricerca di Interesse Nazionale 2005033023. The costs of publication of this article were defrayed in part by the payment of page charges. This article m...

A technique based on liquid chromatography has been developed to facilitate studies of enantioselectivity in penicillin G acylase (PGA)-catalyzed hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues. PGA was covalently immobilized on an aminopropyl monolithic silica support to create an immobilized HPLC-enzyme reactor. Two sets of experimental data were drawn to calculate the enantioselectivity (E) of the kinetically controlled enantiomer-differentiating reaction, the degree of substrate conversion and the enantiomeric excess of the product. The developed enzymatic reactor was coupled through a switching valve to an achiral analytical column for separation and quantitation of the hydrolysis products. The enantiomeric excess was determined off-line on a PGA-chiral stationary phase. In this way, highly precise E values were determined. A computational study related to the hydrolysis of the considered racemic esters was also carried out in order to unambiguously clarify both the substrate specificity and the enantioselectivity displayed by PGA.

No abstract